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X-ray crystallography and mass spectroscopy reveal that the N-lobe of human transferrin expressed in Pichia pastoris is folded correctly but is glycosylated on serine-32.

Bewley MC, Tam BM, Grewal J, He S, Shewry S, Murphy ME, Mason AB, Woodworth RC, Baker EN, MacGillivray RT
Biochemistry 38 2535-41 (1999)
Cited: 15 times
EuropePMC logo PMID: 10029548

Abstract

The ferric form of the N-lobe of human serum transferrin (Fe(III)-hTF/2N) has been expressed at high levels in Pichia pastoris. The Fe(III)-hTF/2N was crystallized in the space group P41212, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 A resolution. This represents only the second P. pastoris-derived protein structure determined to date, and allows the comparison of the structures of recombinant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with serum-derived transferrin. The polypeptide folding pattern is essentially identical in all of the three proteins. Mass spectroscopic analyses of P. pastoris- hTF/2N and proteolytically derived fragments revealed glycosylation of Ser-32 with a single hexose. This represents the first localization of an O-linked glycan in a P. pastoris-derived protein. Because of its distance from the iron-binding site, glycosylation of Ser-32 should not affect the iron-binding properties of hTF/2N expressed in P. pastoris, making this an excellent expression system for the production of hTF/2N.

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Reviews citing this publication (2)

  1. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. Cereghino JL, Cregg JM. FEMS Microbiol Rev 24 45-66 (2000)
  2. Recombinant protein expression in Pichia pastoris. Cregg JM, Cereghino JL, Shi J, Higgins DR. Mol Biotechnol 16 23-52 (2000)

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  1. Transferrin-mediated targeting of bacteriophage HK97 nanoparticles into tumor cells. Huang RK, Steinmetz NF, Fu CY, Manchester M, Johnson JE. Nanomedicine (Lond) 6 55-68 (2011)
  2. Expression and characterization of a low molecular weight recombinant human gelatin: development of a substitute for animal-derived gelatin with superior features. Olsen D, Jiang J, Chang R, Duffy R, Sakaguchi M, Leigh S, Lundgard R, Ju J, Buschman F, Truong-Le V, Pham B, Polarek JW. Protein Expr Purif 40 346-357 (2005)
  3. The role of transferrin in actinide(IV) uptake: comparison with iron(III). Jeanson A, Ferrand M, Funke H, Hennig C, Moisy P, Solari PL, Vidaud C, Den Auwer C. Chemistry 16 1378-1387 (2010)
  4. High-level production of animal-free recombinant transferrin from Saccharomyces cerevisiae. Finnis CJ, Payne T, Hay J, Dodsworth N, Wilkinson D, Morton P, Saxton MJ, Tooth DJ, Evans RW, Goldenberg H, Scheiber-Mojdehkar B, Ternes N, Sleep D. Microb Cell Fact 9 87 (2010)
  5. Crystal structures of two mutants (K206Q, H207E) of the N-lobe of human transferrin with increased affinity for iron. Yang AH, MacGillivray RT, Chen J, Luo Y, Wang Y, Brayer GD, Mason AB, Woodworth RC, Murphy ME. Protein Sci 9 49-52 (2000)
  6. In vitro enzymatic treatment to remove O-linked mannose from intact glycoproteins. Gomathinayagam S, Hamilton SR. Appl Microbiol Biotechnol 98 2545-2554 (2014)
  7. Molecular modeling of human serum transferrin for rationalizing the changes in its physicochemical properties induced by iron binding. Implication of the mechanism of binding to its receptor. Yajima H, Sakajiri T, Kikuchi T, Morita M, Ishii T. J Protein Chem 19 215-223 (2000)
  8. Detailed molecular dynamics simulations of human transferrin provide insights into iron release dynamics at serum and endosomal pH. Abdizadeh H, Atilgan AR, Atilgan C. J Biol Inorg Chem 20 705-718 (2015)
  9. A practical approach for O-linked mannose removal: the use of recombinant lysosomal mannosidase. Hopkins D, Gomathinayagam S, Hamilton SR. Appl Microbiol Biotechnol 99 3913-3927 (2015)
  10. Separation by hydrophobic interaction chromatography and structural determination by mass spectrometry of mannosylated glycoforms of a recombinant transferrin-exendin-4 fusion protein from yeast. Zolodz MD, Herberg JT, Narepekha HE, Raleigh E, Farber MR, Dufield RL, Boyle DM. J Chromatogr A 1217 225-234 (2010)
  11. Partial characterization of the human serum transferrin epitope reactive with the monoclonal antibody TRC-2. Oztürk S, Cirakoglu B, Bermek E. Hybrid Hybridomics 22 165-171 (2003)
  12. Purification and preliminary X-ray studies on hen serotransferrin in apo- and holo-forms. Choudhury D, Thakurta PG, Dasgupta R, Sen U, Biswas S, Chakrabarti C, Dattagupta JK. Biochem Biophys Res Commun 295 125-128 (2002)


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